1. Field of the Invention
This patent application covers methods and devices by which unwanted immune responses against therapeutic proteins may be detected and prevented.
2. Description of the Related Art
Recent advances in genetic engineering and biotechnology have enabled the creation of a number of advanced biotherapeutic drugs, which are usually therapeutic proteins produced by recombinant DNA techniques. These drugs, such as recombinant insulin, interferon, erythropoietin, growth hormone, and the like, have revolutionized modern medicine.
One thing that most modern biotherapeutic drugs have in common is that they often are recombinant DNA cloned versions of natural proteins and protein hormones, or are modified versions of natural proteins. As such, most biotherapeutics have a much higher molecular weight than traditional pharmaceuticals. Additionally, most biotherapeutics tend to be somewhat delicate. Whereas most traditional pharmaceuticals are small molecules, typically robust and resistant to deterioration caused by temperature storage effects, this is not the case for therapeutic proteins. Many biotherapeutic drugs are dependent upon the correct conformation of their protein components. As a result, biotherapeutics are quite temperature sensitive. Many cannot tolerate freezing, because freezing tends to denature proteins and cause the formation of protein aggregates. Many also cannot tolerate storage temperatures much above refrigerator temperatures, since higher temperatures can also promote protein denaturation and formation of protein aggregates. As a result, most modern biotherapeutics must be carefully temperature controlled from the time of manufacture, to the time they are used by the ultimate end user.
The immune system is a complex network of immune system cells, antibodies, cytokines, and other regulatory components designed to detect and destroy foreign (non-self) molecules, while at the same time not attacking native (self) molecules. Thus molecules that naturally occur in the body exhibit immune tolerance. The biological reason for this should be clear, since it is obviously undesirable for the body to attack its own naturally occurring components. Biotherapeutics, by virtue of the fact that they are synthetic analogs of naturally occurring proteins, also are often covered by this same immune tolerance system. Thus medical practice typically assumes that a biotherapeutic that is an analog of a naturally occurring molecule should generally be capable of administration without undue concern for provoking an immune reaction. However as the structure of a biotherapeutic molecule diverges from a native molecule, the possibility of it triggering a “foreign molecule-attack” immune response increases. In particular, the immune system often recognizes protein aggregates as “non-self”, and mounts an immune response against them. Such targets of immune system attack are commonly referred to as “antigens”.
Although modern biotherapeutics have saved countless thousands of lives, and improved the quality of life for countless others, as their use has increased, it has become apparent that the drugs occasionally exhibit unwanted side effects. One of the most distressing side effects is the occasional development of an unwanted immune reaction against the biotherapeutic. This effect is discussed in Rosenberg, Immunogenicity of Biological Therapeutics, A Hierarchy of Concerns, Dev. Biol. Basel, Karger 2003, Vol 112, pp 15-21. These unwanted reactions are sometimes referred to as HADA (human anti-drug antibody) effects.
As discussed in Chamberlain, “Immunogenicity of Therapeutic Proteins”, The Regulatory Review 5:5, August 2002, pp 4-9, such unwanted immune responses can range from mild responses, to very severe responses. In the mild case, which often occurs for diabetics exposed to partially degraded insulin delivered by insulin pumps, antibodies against the biotherapeutic partially neutralize the biotherapeutic, requiring the dose of the biotherapeutic to be increased in order to achieve the same therapeutic effect. Thus in this insulin pump example, affected diabetics require increasingly larger doses of recombinant human insulin in order to achieve good blood glucose control. In other cases, such as has been seen with recombinant erythropoietin (which is a recombinant protein analog to a naturally produced red cell production stimulating hormone), more serious effects can occur. Erythropoietin is often used to stimulate red blood cell production in anemic patients. However antibodies induced by the recombinant erythropoietin biotherapeutic can bind to naturally produced erythropoietin. This can lead to the complete cessation of all subsequent red cell production. This later condition, called “red cell aplasia” can be fatal unless treated by blood transfusion and/or immunosuppressive drugs.
Although vibration, shaking, or light exposure can facilitate the degradation of therapeutic proteins, these effects are usually minor, relative to temperature effects.
It is generally recognized that upon storage, therapeutic proteins degrade by a variety of time-temperature dependent processes, including denaturation, aggregation, oxidation, deamidation, disulfide exchange, and proteolysis. Studies have shown that this time and temperature dependent storage degradation can create immunogenic byproducts, such as protein aggregates, and further have shown that the formation of these immunogenic byproducts is accelerated at higher storage temperatures (Hochuli, “Interferon Immunogenicity: Technical Evaluation of Interferon α2α”, J. Interferon and Cytokine Res. 17 supplement 1: S15-S21, 1997).
Although storing therapeutic proteins at a lower temperature can minimize a number of these processes, other temperature effects often impose a practical lower temperature storage limit. Upon freezing, for example, many proteins undergo conformational changes that can also lead to denaturation, and aggregation. Thus in practice, therapeutic proteins are optimally stored in a rather narrow temperature range, typically 2-8° C.
Curiously, although it is well known that therapeutic proteins are very sensitive to the effects of time and temperature on storage, in general, the biotechnology and pharmaceutical industry has exhibited a profound lack of curiosity as to the effect on biological therapeutics of storage at temperatures other than refrigerated temperature (2-8° C.), room temperature (generally 23-25° C.), or mild elevated temperature (30° C.). There are very few published studies discussing stability outside of these few specified temperature conditions. This lack of curiosity may be due, in part, to the pharmaceutical industry's tradition of working with small molecule drugs, which are typically less temperature sensitive, less immunogenic, and which usually exhibit tolerance to a broad range of storage conditions. In general, the unstated assumption for biotherapeutics has been that it is adequate to simply characterize a therapeutic protein's temperature stability at a few points, and assume that the therapeutic protein will never encounter any other type of temperature conditions after initial shipment.
At present, when pharmaceutical products are shipped, it is standard practice to include temperature monitors as shipping indicators. These monitors, such as the HOBO time-temperature data logger produced by Onset Computer Corporation, Pocasset, Mass.; the Monitor In-transit temperature recorder; the TagAlert® and TempTales® monitors, produced by Sensitech Corporation, Beverly Mass.; and others; inform users if the drug has been exposed to temperature extremes during shipment. However after shipment, such monitors are typically removed.
Similarly, it is common practice to store drugs in refrigerators, which when run in a properly managed health care practitioner setting, will also be monitored and controlled. Normally, however, drugs are stored in more than one refrigerator during their storage lifetime, and this is where problems can occur.
Note that at present, the cold chain between the manufacturer and the ultimate end user has many interface boundaries. At these boundaries, time-temperature monitoring by one system ends, and monitoring by a different system begins. The time and temperature conditions in the boundary between these different systems is usually not monitored or tracked.
Clearly, it is unrealistic to assume that in all steps and interface regions of the cold chain between the pharmaceutical manufacturer and the ultimate use by the health care practitioner or patient, all protein therapeutics will always be carefully temperature controlled. Other areas of medicine do not make such optimistic assumptions. In medical diagnostics, for example, manufacturers and regulators assume that recommended storage and handling conditions may, in fact, be violated. As a result, diagnostics manufacturers and regulators often require that medical diagnostic products incorporate one or more controls or detection methodologies to detect if the diagnostic's recommended storage and handling conditions have been violated. Such approaches are taught by U.S. Pat. No. 6,629,057, and other technology. In this respect, the disparity of practice between the medical diagnostics industry, and the biotherapeutic industry, is quite large.
One explanation for the difference in practice between the medical diagnostics industry and the biotherapeutic industry is ease of quantitation. Medical diagnostics are designed to rapidly convey large quantities of precise numeric information as to their operating condition. Thus problems can be quickly and easily detected. By contrast, biotherapeutics are more difficult to assay, and immunogenicity assays are particularly difficult. However given the now large number of cases in which immunological complications of protein biotherapeutics have been reported, it is clear that these issues need to be addressed.
Consider, for example, the consequences of improper storage conditions on three different products: the first is a food product, the second is a medical diagnostic, and the third is a biotherapeutic protein. In the first case, customers will quickly detect food degradation, either through “off” taste, or possibly food sickness, and the improper storage will be quickly discovered and corrected. In the second case of a medical diagnostic product, the improper storage will also be quickly detected when lab operators run controls, and obtain aberrant answers. Here too, improper storage will be quickly discovered and corrected. However in the third case of a therapeutic protein, the results may be quite different. On a somewhat random basis that may correlate with shipment or storage history, but which will usually not correlate with specific manufacturing lot numbers, certain patients may develop inexplicable immune reactions against the therapeutic protein. This will typically occur many months after the fact. Given the large time lag, difficulty of detection, and the random nature of improper storage conditions, the cause may never be discovered. Yet at the same time, the consequences may be severe. A therapeutic protein pharmaceutical product, or indeed an entire class of therapeutic protein pharmaceuticals, may be subject to regulatory delay or outright recall, affecting the medical status and prognosis of thousands of patients worldwide.
Whether a potentially antigenic therapeutic protein proceeds to produce a clinically unacceptable immune response in a patient depends upon a number of additional factors. Patients differ in their genetic makeup, with some patients tending to be antigen “responders”, and some tending to be antigen “non responders”. Additionally, the route of administration of the antigen may play a role. Mounting an immune response generally takes time. Therapeutic proteins administered in a localized depot, such as by subcutaneous injection, which slowly produces a higher localized level of antigen, may produce a higher immune response than therapeutic proteins administered by an intravenous route. Although differences in patient genetic makeup and route of administration will clearly have an impact on the development of an unacceptable immune response, clearly a key strategy is to simply avoid using potentially antigenic therapeutics in the first place.
Currently, the biotechnology industry expends a great amount of effort in optimizing the chemistry of biotherapeutics, with the goal of minimizing immunogenicity. These efforts include humanizing monoclonal antibodies, modifying the structure of the biotherapeutic proteins, and optimizing the pH, buffer, and carrier molecules that help preserve the original biotherapeutic shape and structure. However in contrast to this extensive amount of effort to optimize biotherapeutic chemistry, a relatively small amount of effort is devoted to monitoring the storage conditions that can cause chemical modifications and antigen formation upon prolonged biotherapeutic storage.
In medical diagnostics, and in many other areas, causes of failure are often analyzed by FMEA (Failure Modes Effects Analysis). This type of analysis allows failure modes to be numerically ranked in order of importance, based upon the severity of the failure, the frequency of occurrence of the failure, and the ability to detect the failure in a timely manner. More severe failures are given a high numeric first coefficient, more frequent failures are given a high numeric second coefficient, and hard to detect failures are given a high numeric third coefficient. Easy to detect failures are generally given a low numeric rating, since failures that can be easily detected can then usually be counteracted quickly. The three coefficients are then multiplied, and the magnitude of the resulting FMEA rating is used as a guide to determine the order and priority in which failure modes should be addressed. Higher FMEA ratings are more urgent, and are generally given a higher priority for subsequent corrective action.
FMEA analysis can be used to examine the three examples of improper storage conditions discussed previously. The first example, improper food storage, although important, would generally be given a medium FMEA priority because the failure is usually simply customer dissatisfaction or gastric distress, and the ability to detect the failure is high. Improper medical diagnostics storage might be given a somewhat higher priority, due to the fact that the impact severity, possible misdiagnosis of a patient, is often quite high. However since control tests are mandated, and frequently performed, the detectability is also high, and the good detectability FMEA coefficient reduces the overall FMEA ranking. By contrast, improper shipment or storage of a protein therapeutic will typically generate a very high FMEA score. The failure mode, possible patient adverse reaction to the drug, possible death, and possible recall of an otherwise promising therapeutic, is extremely severe. At the same time, using current practice, a number of storage condition failures are often difficult or impossible to detect, due to lack of appropriate devices to continually monitor the material at all steps of the cold chain. This combination of high impact and low detectability is quite undesirable. As the frequency of such events increases, the subsequent FMEA ranking may get very high.
At present, pharmaceutical manufacturers are primarily focused on reducing the severity and frequency portion of the FMEA analysis by employing chemical strategies intended to reduce the potential antigenicity of the therapeutic proteins. Although this effort is justified and commendable, FMEA analysis shows that there is another way to reduce risk. This is by improving the detectability of the failure. Health care practitioners or patients who are aware that a particular vial of therapeutic protein has a potential immunogenicity issue due to improper storage or handling can simply avoid using that particular vial. This can be done by incorporating monitoring means with the vial that stay with the vial throughout the cold chain, and that can warn the user about potential immunogenicity issues. Although traditionally, limitations in sensor technology have made such efforts technically or economically infeasible, the rapid advance in modern low cost electronics, instrumentation and detection chemistry, as well as the comparatively high economic value of each vial of therapeutic protein, now make such efforts feasible.